come on, i posed this thread some time ago, but no one answerd me my question:

"so i have the question if in case of macromolecular synthesis. the cultivation of a bacterium lets say staph aureus in this experiment is denoted in many protocols in phosphate buffer...but when they do not grow in the lag phase how can tritium labeled -thymidine or uridine or leucine -be incorporate?"

High Macromolecular Synthesis
with Low Metabolic Cost in
Antarctic Sea Urchin Embryos
Adam G. Marsh,1* Robert E. Maxson Jr.,2 Donal T. Manahan1†
Assessing the energy costs of development in extreme environments is important
for understanding how organisms can exist at the margins of the
biosphere. Macromolecular turnover rates of RNA and protein were measured
at Ð1.5¡C during early development of an Antarctic sea urchin. Contrary to
expectations of low synthesis with low metabolism at low temperatures, protein
and RNA synthesis rates exhibited temperature compensation and were
equivalent to rates in temperate sea urchin embryos. High protein metabolism
with a low metabolic rate is energetically possible in this Antarctic sea urchin
because the energy cost of protein turnover, 0.45 joules per milligram of
protein, is 1/25th the values reported for other animals.
In the cold waters of Antarctica, embryos and
larvae of marine invertebrates have extended
developmental periods (several months to a
year) and low metabolic rates (1). The effects
of limited food and cold temperature are considered
major selective forces resulting in
slow developmental processes in polar seas
(2). The relation between development, macromolecular
synthesis, and energy utilization
is critical for understanding the physiological
mechanisms that determine larval survival in
species inhabiting these extreme environments.
Protein biosynthesis is a major determinant
of an organism’s total energy budget
(about 30%) (3), and studies of temperature
effects on metabolism have led to the suggestion
that changes in protein turnover are an
evolutionary component of metabolic temperature
adaptation (4).
We measured protein synthesis and turnover
during development of the Antarctic sea
urchin Sterechinus neumayeri at –1.5°C by
quantifying rates of incorporation of
[14C]alanine and [14C]leucine into protein.
High-performance liquid chromatography
was used to measure free amino acid pools of
alanine and leucine for calculations of the
intracellular specific activity (5, 6). Gasphase
acid hydrolysis was used to quantify
the mole percent of alanine (7.0% 6 0.2 SD)
and leucine (10.8% 6 0.3 SD) in proteins of
S. neumayeri for calculations of the total
mass of synthesized protein from the amino
acid incorporation rates (7).
Metabolic rates for embryos of S. neumayeri
were low, increasing from only 2 to
12 pmol O2 hour21 per individual during
development to the four-arm, pluteus larval
stage (22 days of development) (8). Once this
pluteus larval stage was reached, respiration
rates stabilized between 13 and 16 pmol O2
hour21 per individual to day 47 (the time
period studied). Protein synthesis rates were
about 5 ng hour21 except during gastrulation
(day 12), when a rapid increase in protein
synthesis was evident (15 to 25 ng hour21;
Fig. 1A) (9). Once the pluteus larval stage
was reached (day 22), further development to
day 50 resulted in a decline in protein synthesis
rates to 0.2 to 0.7 ng hour21. Measurements
of protein synthesis using both alanine
and leucine as tracers were equivalent.
During embryogenesis and early development
of unfed larvae of S. neumayeri, measured
rates of protein synthesis were equivalent
to rates of protein turnover, because there
was no increase in protein mass of the embryos
or larvae (10). The fractional rates of
protein turnover for S. neumayeri were calculated
by expressing rates of synthesis as a
percentage of total protein content during
development. These fractional rates ( percentage
of total body protein turned over per
hour) were 2.2% for blastulae (11), increased
during gastrulation (10% per hour), and were
then low in pluteus larvae (,1% per hour).
Reports of protein turnover for blastula stages
of some well-studied temperate species of sea
urchins provide data for a comparison of
fractional rates of protein turnover ( percent
per hour): S. neumayeri, 2.2% (–1.5°C);
Strongylocentrotus purpuratus, 1.1% (16°C)
(12); Lytechinus pictus, 1.0% (19°C) (13);
and Arbacia punctulata, 1.9% (25°C) (14).
These rates were equivalent despite large differences
in environmental temperatures. The
fractional rate of protein turnover in embryos
of S. neumayeri at –1.5°C exhibits temperature
compensation (2% per hour), despite a
thermal gradient that should theoretically result
in a physiological rate reduction in protein
turnover to one-fourth to one-sixth of the
values shown for temperate sea urchin embryos
(using a conservative Q10 5 2, a common
parameter used to adjust physiological
rate measurements between different temperatures;
Fig. 1B). We conclude that embryos
of S. neumayeri demonstrate full temperature
compensation for rates of protein turnover.
To address a potential mechanism for the
high protein turnover rates at –1.5°C , we measured
the amount of whole-cell RNA and
poly(A1) RNA in eggs and embryos of S.
neumayeri and, for comparison, in S. purpuratus.
Both whole-cell RNA and poly(A1) RNA
fractions were substantially higher in hatching
1Department of Biological Sciences, University of
Southern California, Los Angeles, CA 90089, USA.
2Department of Biochemistry and Molecular Biology,
USC/Norris Hospital Institute, University of Southern
California School of Medicine, Los Angeles, CA 90033,
USA.
*Present address: College of Marine Studies, University
of Delaware, Lewes, DE 19958, USA.
†To whom correspondence should be addressed. Email:
[email protected]Fig. 1. Protein turnover rates during development
of S. neumayeri. (A) Protein turnover
rates (that is, synthesis rates in embryos and
larvae of S. neumayeri ) during development
were calculated from the free amino acid speci
Þc activity of a radiolabel, the mole percent
amino acid composition, and the rates of labeled
amino acid incorporation into protein.
Open symbols represent four different cultures
for which alanine was used as the radiolabeled
tracer; leucine was also used for corroboration
of rates (gray triangles) on one of those cultures.
All points are plotted as means 6 1 SEM
(n 5 5). (B) Fractional rates of protein turnover
( per unit egg protein mass; percent per hour)
for different species of sea urchin embryos at
the blastula stage. The temperatures at which
these measurements were made are indicated
above the solid bars. The hatch bars indicate
the turnover rates at Ð1.5¡C as directly measured
in S. neumayeri (Sn) and estimated by
Q10 extrapolations (using a conservative value
of 2.0) in the temperate urchins Arbacia
punctulata (Ap), Lytechinus pictus (Lp), and
Strongylocentrotus purpuratus (Sp).
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blastulae of S. neumayeri than those of S. purpuratus.
Whereas hatching blastulae of S. purpuratus
contain a constant 3 ng of whole-cell
RNA (15) and 69 6 1.2 pg of poly(A1) RNA
(our data) during early development, S. neumayeri
embryos show a large increase in
poly(A1) RNA levels (Fig. 2A), and at the
blastulae stage, they contain as much as 115 6
11 ng of whole-cell and 8165 6 441 pg of
poly(A1) RNA (per embryo). Normalized to
embryo volume, S. neumayeri had approximately
10 times the cellular concentration of
poly(A1) RNA (2.67 fg mRNA mm23) as did
S. purpuratus (0.25 fg mRNA mm23) (15).
Elevated levels of poly(A1) RNA and wholecell
RNA may in part explain the mechanistic
basis for the high rates of protein turnover in
embryos of S. neumayeri.
Greater levels of poly(A1) RNA in S.
neumayeri may result from increased synthesis
rates and/or reduced degradation rates.
Measurements of absolute rates of synthesis
of total RNA in S. neumayeri ( picograms
RNA per hour per cell) evidenced temperature
compensation and were equivalent to
rates in S. purpuratus (15) and L. pictus (16),
despite large differences in environmental
temperatures (Fig. 2B). Even more noteworthy,
the synthesis rates of whole-cell
poly(A1) mRNA were much higher in S.
neumayeri (0.14 pg mRNA hour21 per cell)
when compared with embryos of L. pictus
and S. purpuratus (0.04 and 0.03 pg mRNA
hour21 per cell, respectively), even without a
temperature correction (Fig. 2C). In addition,
the percentage of total RNA synthesis allocated
to mRNA production was higher in S.
neumayeri (42% versus 9% of total RNA
synthesis; compare parts B and C of Fig. 2).
The turnover of poly(A1) mRNA was calculated
from a kinetic analysis of the radiolabel
incorporation rates (17) and revealed a halflife
of 4.1 hours in S. neumayeri. A similar
analysis for L. pictus from published data
(16) produced a near-equivalent turnover
time of 4.3 hours. Thus, the 10-fold elevation
of poly(A1) mRNA per unit volume in S.
neumayeri relative to S. purpuratus (Fig. 2A)
most likely results from an increased synthesis
rate of mRNA and not a reduced degradation
rate at low temperatures.
High rates of macromolecular turnover
with low rates of respiration in S. neumayeri
pose a physiological paradox: a 2% per hour
protein turnover rate would be energetically
impossible if metabolic energy costs of protein
metabolism were the same at –1.5°C as
has been reported in other animals. The aerobic
cost of protein turnover can be estimated
from a linear regression of total respiration
rates against protein synthesis rates, an approach
used routinely in metabolic studies
(18). This cost includes the energy utilization
of all cellular activities involved in protein
turnover, including RNA synthesis and processing.
For late embryos and larval stages of
S. neumayeri (after gastrulation, day 17 to
day 47) the regression revealed a low energy
cost of protein turnover (Fig. 3A). The slope
of this regression line was 0.94 (6 0.14 SE)
pmol O2 hour21 respired for 1 ng hour21 of
protein turnover (r 2 5 0.802, n 5 13), which
is equivalent to 0.45 J mg21 protein (at 484
kJ mol21 O2). A synthesis cost of 0.45 J
mg21 protein in S. neumayeri is about 1/25th
of values reported for a variety of other animals
calculated by using a similar approach
[e.g., the mussel Mytilus edulis, 11.4 J mg21
(19); a cod fish, 8.7 J mg21 (20); mammals,
12.6 J mg21 (21)].
Using a net energy cost of 0.45 J mg21
protein for S. neumayeri, protein synthesis
rates (Fig. 1A) can be converted to the fraction
of total energy metabolism consumed by
protein turnover during development. The
contribution of protein turnover to total metabolic
demand was 53% at the hatching blastula
stage (Fig. 3B). The in vivo metabolic
energy consumption of Na1/K1-ATPase (the
sodium ion pump, another major component
of cellular energy metabolism in animals) has
been measured during development in S. neumayeri
(10). For a hatching blastula, protein
turnover and sodium ion regulation account
for 65% of total metabolism (Fig. 3B), which
is consistent with what is known about metabolic
energy partitioning in other animals
Fig. 2. Amount of RNA and rates of synthesis
in S. neumayeri. (A) Amounts of poly(A1)
mRNA are presented for S. neumayeri and S.
purpuratus (mean 6 1 SD) during early development
for a zygote (egg), and blastula
(blast) and hatching blastula (hatch) stages.
(B) Synthesis rates of whole-cell, total RNA
( picograms per hour per cell) for different
species of sea urchin embryos at the blastula
stage. The temperatures at which these measurements
were made are given below. The
hatched bars indicate the synthesis rates at
Ð1.5¡C as directly measured in S. neumayeri
(Sn, Ð1.5¡C) and estimated by Q10 calculations
(using a conservative value of 2.0) in
the temperate urchins Lytechinus pictus (Lp,
19¡C) and S. purpuratus (Sp, 16¡C) [from
published values (13, 14)]. (C) Synthesis rates
of whole-cell, poly(A1) mRNA (mRNA, picograms
per hour per cell) for different species
of sea urchin embryos at the blastula stage
(Lp, 19¡C; Sp, 16¡C). Errors for the S. neumayeri
synthesis rates in (B) and (C) were
calculated but are not shown on the graphÑ
the standard error of the regression coefÞ-
cients that were used to calculate these net
synthesis rates was approximately 6 20%.
Fig. 3. Energetics of protein metabolism in
embryos and larvae of S. neumayeri. (A) The
energy costs of protein turnover rates during
development of S. neumayeri were calculated
from a regression of respiration and protein
synthesis rates. A linear relation between respiration
and protein synthesis was evident after
day 17 of development, the transition from a
late prism-stage embryo to early pluteus-stage
larva (see Fig. 1). Symbols correspond to the
same cultures as in Fig. 1. Protein turnover was
measured in larvae with both alanine (open
symbols) and leucine (gray triangles) as the
radiolabeled amino acid tracers. The data (n 5
13) include some previously measured respiration
rates [(open triangles (27)]; other symbols
represent independent measurements made on
additional cultures. (B) The fraction of total
metabolic energy expenditure accounted for by
protein metabolism in S. neumayeri. Energy
consumed by protein metabolism was calculated
from the ratio of respiration to protein
turnover [0.45 J mg21 protein; (A)] and then
expressed as a percentage of total metabolic
energy expenditure. Energy consumption of the
Na1 pump has been published elsewhere (10).
R E P O R T S
http://www.sciencemag.org SCIENCE VOL 291 9 MARCH 2001 1951
(22). Once a larval stage is reached (day 22),
protein metabolism had decreased to 30% of
total metabolism (23) and then further declined
to 1% for a larva at day 50 (Fig. 3B,
right-side of pie chart). At this point in larval
development, the sodium pump consumes a
very large fraction of total metabolic energy
(80%) (10), and a reduction in the cost of
protein turnover is necessary to accommodate
the sodium pump’s demand for cellular energy,
given the low metabolic rates of these
embryos and larvae.
A relative increase in the rates of mRNA
synthesis and protein turnover at –1.5°C is energetically
possible in S. neumayeri, because
the cost of protein metabolism is very low.
Indeed, the value we report is lower than has
been reported for any other animal. The increase
in poly(A1) mRNA synthesis can provide
a proximate explanation for the unexpectedly
high rate of protein turnover in this Antarctic
animal. The thermodynamic bases remain
to be elucidated for such energy efficiency
of protein turnover at low temperatures. Further
analyses of the processes underlying the greater
energy efficiency in protein metabolism may
uncover novel mechanisms of biochemical adaptations
and lead to a better understanding of
metabolic diversity in organisms inhabiting extreme
polar environments.
References and Notes
1. F. M. Shilling, D. T. Manahan, Biol. Bull. 187, 398 (1994).
2. A. Clarke, Oceanogr. Mar. Biol. Annu. Rev. 21, 341
(1983).
3. W. Weiser, Biol. Rev. 68, 1 (1994).
4. H. A. Thieringer, P. G. Jones, M. Inouye, Bioessays 20,
49 (1998).
5. J. R. Welborn, D. T. Manahan, J. Exp. Biol. 198, 1791
(1995).
6. Early work on protein synthesis rates in sea urchin
embryos has demonstrated a rapid equilibration between
the intracellular free amino acid pool and the
charged tRNA pool (24).
7. Amino acid composition of proteins was examined
during development in S. neumayeri by gas-phase
acid hydrolysis and high-performance liquid chromatography
quantiÞcation of constituent amino acids
(5, 25). A trichloroacetic acid precipitation step was
used to isolate protein from whole embryos and
larvae for the analyses. Seven different developmental
time points were measured between fertilization
and the Þrst larval stage (day 22).
8. Respiration rates were measured using 1-ml glass respiration
vials for end-point measurements of oxygen
tension using a polarographic oxygen sensor (26, 27).
Six vials with 50 to 200 embryos or larvae were measured
at each experimental point by sealing the vials
and incubating them for 8 hours at 21.5¡C. Oxygen
tension was then measured by injecting 500 ml of the
seawater from a vial into a 50-ml microcell maintained
at21.5¡C. This technique was speciÞcally optimized for
use with S. neumayeri embryos and larvae and crosschecked
with four other independent methods for measuring
oxygen consumption rates.
9. Protein synthesis rates were measured during 90-min
time-course experiments of radiolabel incorporation
into trichloroacetic acidÐprecipitable protein (28).
During embryogenesis, rates were measured on four
separate cultures (started from different parents at
different times), and two of these were further used
for studies during later larval development. For all
experiments, 12,000 individuals were placed in 12 ml
of sterile Þltered seawater (0.2 mm) with 70 mCi of
[14C]alanine. Individuals were incubated at 21.5¡C
with the radiolabel, and 500-ml aliquots were removed
at 15-min intervals for measurements of both
the protein incorporation and the speciÞc activity of
[14C]alanine in the free amino acid pool. Corroborative
measurements were also made using
[14C]leucine.
10. P. K. K. Leong, D. T. Manahan, J. Exp. Biol. 202, 2051
(1999).
11. The number of cells in the blastulae stage has been
measured previously, 2152 cells (27), allowing a calculation
here of a cell-speciÞc rate of protein synthesis
of 1.49 pg protein per hour per cell.
12. A. S. Goustin, F. H. Wilt, Dev. Biol. 82, 32 (1981).
13. W. E. Berg, D. H. Mertes, Exp. Cell Res. 60, 218
(1970).
14. B. J. Fry, P. R. Gross, Dev. Biol. 21, 125 (1970).
15. E. H. Davidson, Gene Activity in Early Development
(Academic Press, Orlando, FL, ed. 3, 1986).
16. R. S. Wu, F. H. Wilt, Dev. Biol. 41, 352 (1974).
17. M. Ito, J. Bell, G. Lyons, R. Maxson, Dev. Biol. 129, 147
(1988).
18. D. F. Houlihan, in Advances in Comparative and Environmental
Physiology, R. Gilles, Ed. (Springer-Verlag,
Berlin, 1991), vol. 7, pp. 1Ð43.
19. A. J. Hawkins, J. Widdows, B. L. Bayne, Physiol. Zool.
62, 745 (1989).
20. A. R. Lyndon, D. F. Houlihan, S. J. Hall, Arch. Int.
Physiol. Biochem. 97, C31 (1989).
21. P. J. Reeds, Anim. Prod. 45, 149 (1987).
22. W. G. Siems, H. Schmidt, S. Gruner, M. Jakstadt, Cell
Biochem. Funct. 10, 61 (1992).
23. A. G. Marsh, R. E. Maxson Jr., D. T. Manahan, data not
shown.
24. J. C. Reiger, F. C. Kafatos, Dev. Biol. 57, 270 (1977).
25. R. Heinrikson, S. Meredith, Anal. Biochem. 136, 65
(1984).
26. A. G. Marsh, D. T. Manahan, Mar. Ecol. Prog. Ser. 184,
1 (1999).
27. A. G. Marsh, P. K. K. Leong, D. T. Manahan, J. Exp. Biol.
202, 2041 (1999).
28. J. Vavra, D. T. Manahan, Biol. Bull. 196, 177 (1999).
29. This work was supported by the National Science
Foundation OPP-9420803. We thank T. Hamilton for
technical assistance with the experiments and chromatographic
analyses. This paper is dedicated to the
late Catherine Manahan.
4 October 2000; accepted 30 January 2001
Published online 15 February 2001;
10.1126/science.1056341
Include this information when citing this paper.
A Short Duration of the
Cretaceous-Tertiary Boundary
Event: Evidence from
Extraterrestrial Helium-3
S. Mukhopadhyay,1* K. A. Farley,1 A. Montanari2
Analyses of marine carbonates through the interval 63.9 to 65.4 million years
ago indicate a near-constant ßux of extraterrestrial helium-3, a tracer of the
accretion rate of interplanetary dust to Earth. This observation indicates that
the bolide associated with the Cretaceous-Tertiary (K-T) extinction event was
not accompanied by enhanced solar system dustiness and so could not have
been a member of a comet shower. The use of helium-3 as a constant-ßux proxy
of sedimentation rate implies deposition of the K-T boundary clay in (1062)3
103 years, precluding the possibility of a long hiatus at the boundary and
requiring extremely rapid faunal turnover.
The K-T boundary at 65 million years ago
(Ma) records a major mass-extinction event
and, though the occurrence of an extraterrestrial
impact (1, 2) is widely accepted, the
nature of the impactor and its role in the K-T
mass extinction is debated. Possible candidates
for the impactor are a single asteroid or
comet (1–3) or a member of a comet shower
(4). An extraterrestrial impact would have
severely perturbed Earth’s ecosystems and
climate by injecting large quantities of dust
(1) and climatically active gases (5) into the
atmosphere. An alternative hypothesis to explain
the biotic calamity invokes voluminous
volcanism (6). Recent work (7) suggests that
most of the Deccan Traps flood basalts were
erupted in a ,1-million-year (My) interval
coincident with the K-T boundary. The global
environmental effects from extensive volcanism
could be similar to the effects from a
large impact (6), but the time scale of the two
processes would be different. The perturbation
on climate and ecosystems from an impact
would be geologically instantaneous, but
the effects from volcanism would be spread
over at least a few hundred thousand years.
The K-T boundary clay is a distinctive
bed, typically a few cm thick, that separates
sedimentary rocks of the Cretaceous from
those of the Tertiary. Knowledge of the deposition
interval of the clay would provide
important insights into the cause(s) and rates
of mass extinction and climate change at the
boundary, but most geochronologic tools are
inadequate for this purpose. Estimates of this
time interval are based on the assumption that
the K-T clay was deposited at the same rate
1Division of Geological and Planetary Sciences, California
Institute of Technology, Pasadena, CA 91125,
USA. 2Osservatorio Geologico di Coldigioco, 62020
Frontale di Apiro, Italy.
*To whom correspondence should be addressed. Email:
[email protected]R E P O R T S
9 MARCH 2001 VOL 291 SCIENCE http://www.sciencemag.org 1952